Consuming a healthy diet is one of the controllable factors that influences the development or severity of multiple chronic diseases, including metabolic, inflammatory, and neoplastic disorders. Diets high in cruciferous vegetables consistently show a decreased incidence of colorectal cancer and cardiovascular disease. Understanding the dynamic interrelationships that exist between bioactive food components in broccoli and human disease prevention is recognized as a high priority. A collection of 125 near-isogenic broccoli lines (Brassica oleracea subsp. italica) previously developed by our group, represents a unique tool for studying effects of multiple dietary bioactive components by utilizing physiologically relevant concentrations of the agents and eliminating experimental noise associated with food matrix effects. By using broccoli near-isogenic lines, we successfully observed direct nutrient-nutrient or nutrient-disease interactions that vary significantly (order of magnitude) in their ability to suppress inflammation in RAW 264.7 macrophages or inhibit proliferation of HT-29 colorectal cancer cells. When combined with quantitative trait loci (QTL) or genome-wide association (GWAS) studies, this approach allowed for direct mapping of human health-modifying regions (HMRs) in the broccoli genome – even if the interacting bioactive metabolites, or their respective biosynthetic and genetic regulatory pathways, were unknown. Our findings suggested that 3 health-modifying regions (HMRs) on 2 chromosomes were sufficient to explain over 52% of the variation in health-promoting activity observed among broccoli varieties tested. Within these regions, known gene sequences were assessed for relevance to synthesis or regulation of bioactive metabolites to identify candidate genes and advance breeding programs aimed at targeted improvement of broccoli nutritional value.